Qualitative and quantitative detection of very small amounts of substances contained in biological and non-biological samples are commonly made in clinical examinations, diagnoses, analyses, self care, etc.
In such detection, an analyte in a sample is usually reacted with an indicator substance that reacts specifically with the analyte. The reaction product(s) or reaction composite(s) produced as a result of the reaction is/are detected by methods such as colorimetry (absorptiometric analysis), fluorometry, radiometry, chemiluminescent assay, electrochemical method, impedance method, quartz crystal microbalance method (QCM), surface plasmon resonance method (SPR), and thermal lens effect detection, to detect or quantify the analyte. A common indicator substance used in the detection or quantification of an analyte is 5-methylphenazinium methyl sulfate (chemical formula: C14H14N2O4S, hereinafter abbreviated as PMS). PMS reduced by the reaction with a specific analyte is detected by an electrochemical method or colorimetry to detect or quantify the analyte.
However, PMS is unstable and easily oxidized by light in an aqueous solution. Thus, it is known that the storage of such reagent solutions is difficult. To avoid the problem of storage, 1-methoxy-5-methylphenazinium methyl sulfate (chemical formula: C15H16N2O5S, hereinafter abbreviated as M-PMS), a derivative of PMS, was developed. M-PMS is a compound in which a methoxy group is introduced into the first position of PMS, and is very stable with respect to light.
The measurement of the concentration of creatinine contained in a sample is important in the fields of clinical chemistry and analytical chemistry. Since creatinine is a product of the endogenous metabolism of muscle, it is known that the amount of creatinine in urine reflects total muscle mass. Hence, it is believed that the amount of creatinine excretion in the urine of each individual in a day is usually constant and does not vary from day to day. As such, the amount of urinary creatinine may be used as a measure of the thickness of excreted urine. Also, the amount of creatinine in urine and blood increases/decreases due to uremia or decreased renal function. Thus, the measurement of the amount of creatinine in urine or blood permits determination of the presence or absence of uremia or decreased renal function.
A known method for measuring creatinine concentration is a method based on Jaffe reaction using an alkaline picrate solution. According to this method, the orange-red product formed by the reaction between picric acid and creatinine is spectroscopically measured (see, for example, PTL 1).
Another known method for measuring creatinine concentration is a method using an enzyme that reacts specifically with creatinine. An example of such an enzymatic method is a method of decomposing creatinine using creatinine deiminase. According to this method, the amount of ammonia produced by the decomposition of creatinine is measured based on the change in pH, potential, and the like to determine creatinine concentration (see, for example, PTL 2).
Another enzymatic method is a method of measuring creatinine concentration by carrying out the following reactions of formulas (1) to (3):Creatinine+Water→Creatine  (1)Creatine+Water→Sarcosine+Urea  (2)Sarcosine+Water+Oxygen→Glycine+Formaldehyde+Hydrogen Peroxide  (3)
The enzymes used to catalyze the reactions of formulas (1) to (3) are creatinine amidohydrolase (creatininase), creatine amidinohydrolase (creatinase), and sarcosine oxidase or sarcosine dehydrogenase, respectively. Creatinine is quantified, for example, by a method of using a leuco pigment or the Trinder reagent together with a peroxidase to cause the hydrogen peroxide produced in formula (3) to give a color for spectroscopic quantification (see, for example, PTL 3). Also, another creatinine quantification method is a method of electrochemically oxidizing the hydrogen peroxide produced in formula (3) at an electrode to cause a current to flow and quantifying creatinine from the current (see, for example, PTLs 4 and 5).
Further, still another enzymatic method is a method of quantifying creatinine by carrying out the reactions of formula (1) and formula (2) and additionally carrying out the reaction between sarcosine and an electron mediator instead of the reaction of formula (3) (see, for example, PTLs 6 and 7).
PTL 6 discloses a creatinine biosensor including at least a pair of a working electrode and a counter electrode on a substrate, wherein a reagent solution is dried on the electrodes or on the substrate near the electrodes to immobilize the reagent. The reagent solution is prepared by dissolving creatininase, creatinase, sarcosine oxidase, and potassium ferricyanide (electron mediator) in a buffer solution of pH 7 to 8.5. It is also disclosed that a buffer solution pH of less than 7 or greater than 8.5 is not preferable since the enzyme activity decreases.
PTL 7 discloses quantifying creatinine by colorimetry or an electrochemical detection method using sarcosine oxidase and a mediator (electron mediator) encapsulated in cyclodextrin. Specifically, PTL 7 cites α-naphthoquinone (1,4-naphthoquinone) as an example of a mediator encapsulated in cyclodextrin, but discloses that mediators are not suitable for the enzymatic measurement of creatinine if they are not encapsulated in cyclodextrin.
Also, still another enzymatic method is a method of spectroscopically quantifying creatinine by carrying out the reactions of formula (1) and formula (2) and additionally carrying out the reaction between sarcosine and a tetrazolium indicator instead of the reaction of formula (3) (see, for example, PTL 8). PTL 8 discloses that the composition for creatinine quantification comprises a reagent mixture of creatinine hydrolase, creatine amidinohydrolase, sarcosine dehydrogenase, thiazolyl blue serving as the tetrazolium indicator, and potassium phosphate of pH 7.5.
Also, still another enzymatic method is a method of converting creatinine to glycine and formaldehyde by use of creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine dehydrogenase, causing the produced formaldehyde to give a color with the aid of a color reagent, and quantifying creatinine from the absorbance (see, for example, PTL 9). PTL 9 discloses using a phosphate buffer solution of pH 7.5 and potassium ferricyanide serving as a reaction accelerator for promoting the formation of formaldehyde in addition to creatinine amidohydrolase, creatine amidinohydrolase, sarcosine dehydrogenase, and the color reagent.
Also, still another enzymatic method is a method of quantifying creatinine using an electrode on which a polymer that catalyzes the hydrolysis of creatinine, sarcosine oxidase, and a mediator are immobilized (see, for example, PTL 10). PTL 10 discloses using, for example, potassium ferricyanide, ferrocene, an osmium derivative, or phenazine methosulfate (PMS) as the mediator.
Still another method of creatinine concentration quantification is a method of using 1,4-naphthoquinone-2-potassium sulfonate (see, for example, PTL 11 and NPLs 1 to 3).